Cervical cancer is the second most common cancer in women worldwide. Papanicolaou (Pap) cytology is an established screening technique that has led to a significant reduction of incidence and mortality in cervical cancer. It is important t detect the clinically significant high-grade cervical intraepithelial neoplasia (HGCIN) that requires further extensive and invasive examination and treatment. However, about 3 million Pap smears in US annually receive a diagnosis of atypical squamous cells of undetermined significance (ASC-US), but HGCIN is confirmed on a subsequent follow-up colposcopy biopsy for only less than 5% of patients receiving this diagnosis. While the emergence of human papillomavirus (HPV) testing has enhanced cervical cancer detection, but only a very small number of HPV-infected patients harbor HGCIN. Due to the low specificity of Pap cytology and HPV testing, substantial unnecessary expenses and efforts have been made to identify these HGCIN patients and over-treatment. Novel biomarkers are crucially necessary to enhance the detection of true high-risk patients, especially in HPV-infected patients and allow more effective cervical cancer screening. The main objective of this application is to identify novel biomarkers to improve the accurate detection of HGCIN and malignancy in patients with ASC-US cytology and/or HPV infection. Our primary aim is to identify novel and clinically applicable optical biomarkers for detecting HGCIN and malignancy based on the analysis of nanoscale structural characteristics. Our recently developed Spatial-domain Low-coherence Quantitative Phase Microscopy (SL-QPM) can detect, with nanoscale sensitivity, sub-cellular structural changes (0.9 nm) in standard cytology and histology slides without any additional modification. We have demonstrated that SL-QPM-derived nuclear nano-architecture biomarkers can unambiguously detect several types of cancer even in cells labeled as normal or indeterminate by expert pathologists that were confirmed at surgery to be cancerous. We have also validated the analytical reproducibility and developed a standard operating protocol for SL-QPM analysis. We hypothesize that atypical and cytologically normal squamous cells from patients with HGCIN will exhibit subtle nano-architectural changes in the cell nuclei that appear invisible to conventional cytology, but can be distinguished using SL-QPM-derived biomarkers. In this project, we will improve the throughput of this technique for routine clinical use and further refine and validate these novel optical biomarkers to detect HGCIN in routine cytology slides from women with ASC-US cytology. Further, a proof-of-concept study will be performed to confirm the accuracy of SL-QPM markers when implemented in a real clinical setting in patients with ASC-US cytology and positive HPV test. Given the cost-effectiveness of SL-QPM and the ease with which it can be integrated into the current clinical workflow (including in community and resource-limited settings), our technique has the potential to significantly prevent the overtreatment of women with such diagnoses, reducing anxiety, invasive procedures, and associated healthcare costs in the process.